Multiple sclerosis (MS) is an inflammatory disease of the central nervous system which involves autoreactive CD4+ T cells of a TH1 and TH17 phenotype. Identifying novel factors that control the inflammatory phenotype of T cells will enable a more complete understanding of T cell regulation and MS pathogenesis. MicroRNAs (miRs), which can negatively regulate multiple target genes, are positioned to act as regulatory nodes in gene networks such as those controlling T cell differentiation and function. MiRs are a class of small non-coding RNAs that negatively regulate post-transcriptional gene expression. MiRs have an established role in the immune system and profiling studies have shown that certain miRs are aberrantly expressed in autoimmune diseases, including MS. Mice deficient in small RNA processing machinery, Dicer, produce more cytokines when their immune cells are activated. Together, these data indicate that miR(s) are central to immune system homeostasis and may act as negative regulators of inflammation. However, the miRs responsible for modulating inflammatory cytokines have not been defined, and the function of specific miRs in CD4+ T cells is the subject of this proposal. Using target prediction algorithms, we found that the miR-29 family (miR-29a, miR-29b, miR-29c) is predicted to target both IFN3, the prototypical TH1 cytokine, as well as T-bet, a T-box transcription factor considered the master regulator of TH1 differentiation. MiR-29 is therefore positioned to modulate multiple developmental stages in TH1 cells, including effector cytokine production and transcriptional programming. We hypothesize that miR-29 modulates CD4+ T cell differentiation and propose to explore the biological consequences of altered miR-29 expression using in vitro TH1-polarized murine and human T cells in specific aim 1. The miR-29 promoters each contain 3 conserved IFN3-activated site (GAS) elements within 5kb of other demonstrated transcription factor binding sites, suggesting miR-29 may be transcriptionally regulated through interferon signaling. We have shown that IFN3 stimulation of naove murine T cells is sufficient to up-regulate miR-29 expression. We propose to extend our findings to IFN2 treatment of MS patients, since both IFN3 and IFN2 signal through the STAT pathway. We hypothesize that miR-29 expression is induced through IFN2 treatment of MS patients, and propose to use a unique collection of PBMC from MS patients pre- and post- IFN2 therapy to analyze changes in miR-29 expression in specific aim 2. These studies could result in the discovery of a novel regulatory network capable of influencing TH cell development and function, ultimately contributing to our understanding of the TH1/TH2 paradigm and IFN2 treatment in MS.